Given that mast cells increase increase at sites of inflammation and are located at surfaces where exposure to invading organisms may occur, we examined the Toll-like receptors expressed by cultured human mast cells and the ability to activate mast cells with TLR ligands. We found that viruses and polyI:C induced IFN-alpha and IFN-beta, in part through TLR-3. Human mast cells also expressed TLR-1, TLR-2, TLR-4, TLR-5, TLR-6, TLR-7, and TLR-9. The TLR ligands LPS, peptidoglycan, and flagellin induced the production of TNF, IL-1-beta, IL-5, and GM-CSF. IFN-alpha production involved nuclear factor-kappaB, p38; and C-Jun NH2-terminal kinase and mitogen-activated protein kinase. IFN-gamma treated human mast cells express FcgammaRI. Such mast cells when exposed to heat-aggregated IgG1 degranulate and subsequently generate PGD2 and LTC4; as well as release IL-3, IL-13, GM-CSF, and TNF-alpha. No mast cell activation was observed with the addition of heat-aggregated IgG2, IgG3, or IgG4. Simultaneous activation of human mast cells with aggregated IgG and C3a led to additive degranulation.